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As far as is known at present, fumigants enter the insect mainly by way of the respiratory system. The entrance to this system in larvae, pupae and adults is through the spiracles, which are situated on the lateral surfaces of the body. The opening and closing of the spiracles are under muscular control. To enter insect eggs, gases diffuse through the shell (chorion) of the egg or through specialized "respiratory channels". It has been shown that some gases may diffuse through the integument of insects, but at present the comparative importance of this route for the entry of fumigants is not known.
It is known that the poisoning of an insect by a fumigant is influenced by the rate of respiration of that insect; any factor that increases the rate of respiration tends to make the insect more susceptible.
The practical significance of the more important factors influencing the toxic action of fumigants is discussed in the following paragraphs.
EFFECT OF TEMPERATURE
The most important environmental factor influencing the action of fumigants on insects is temperature. In the range of normal fumigating temperatures from 10 to 35°C, the concentration of a fumigant required to kill a given stage of an insect species decreases with the rise in temperature. From the purely biological standpoint, this is mainly due to the increased rate of respiration of the insects in response to the rise in temperature (Sun, 1946). Also, as pointed out previously, physical sorption of the fumigant by the material containing the insects is reduced and proportionately more fumigant is available to attack the insects. Therefore, within the range mentioned, conditions for successful fumigation improve as the temperature rises. These conditions are reflected in the schedules for recommended treatments included in this manual.
Low Temperature Fumigation
At temperatures below 10°C, the situation is more complicated. Below this point, increased sorption of the gas by the body of the insect may counterbalance the effects of decrease in respiration, and also the resistance of insects may be weakened by the effects of exposure to low temperatures. With some fumigants, less gas is required to kill certain species as the temperature is raised or lowered on either side of some point at which the insects are most tolerant (Moore, 1936; Peters and Ganter 1935; Bond and Buckland, 1976). However, with others, toxicity to the insects declines as the temperature falls; for example, with methyl bromide there is a moderate decrease in toxicity down to the boiling point and below this temperature effectiveness drops off sharply so that the amount of gas required to kill the insects increases dramatically, as shown in Figure 6.
FIGURE 6. - Mortality (LD'9) of Sitophilus qranarius adults and Tenebroides mauritanicus larvae when exposed to methyl bromide for eight hours at different temperatures.
For the reasons already given in the previous discussion, at lower temperatures sorption of the fumigant by the infested material is increased and more fumigant must be applied to compensate for this. Also, diffusion of a gas is slowed down in relation to reduction in temperature.
Prefumination and Postfumioation Temperatures
It is important to bear in mind that the results of a fumigation may be influenced not only by the temperature prevailing during the treatment, but also by the temperatures at which the insects are kept before and after treatment.
If the insects have been kept in a cool environment, their metabolic rate will be low. If they are immediately fumigated at a higher temperature, their physiological activity may still be influenced by their previous history, and the uptake of the poison may not be as great as if they had been kept at the temperature of fumigation for a long time previous to treatment (Pradhan and Govindan, 1953-54). These phenomena can be of practical significance, particularly for certain species of insects that may go into a state known as diapause (see Howe (1962) for description of diapause and list of species involved). For insects in this state, tolerance to some fumigants, e.g. methyl bromide and phosphine, may be several times greater than for non-diapausing insects (Bell, 1977 a,b). For other species not in diapause, toxicity is usually found to be closely dependent on the temperature of the fumigation (Bond, 1975; Bond and Buckland, 1976).
A fumigator must have some knowledge of the previous history of the infested material as well as the species to be treated if he or she is to apply the recommended fumigation treatments most effectively. In all treatments, the material should be warmed to the treatment temperature for several hours to bring the insects to corresponding physiological activity before fumigating. If species disposed to the state of diapause are present (e.g. some members of the order Lepidoptera and the families Dermestidae and Ptinidae of the order Coleoptera) the dosage and exposure applied should be increased to a level that will kill the most tolerant insects.
Under experimental conditions, variations in postfumigation temperatures have been observed to influence insect mortalities, but the effects are more complex than those observed in the study of prefumigation temperatures. However, the net contribution of the postfumigation temperature effects would not be of sufficient importance in practice to influence the results of the procedures recommended in this manual. Reference to the papers of Sun (1946) and of Pradhan and Govindan (195354) should be made by those wishing to pursue this aspect of the subject.
Summary of Temperature Effects
From the foregoing discussion it is clear that temperature has farreaching effects on all the factors governing the successful outcome of fumigation. In order to clarify the significance of these effects they may be summarized as follows:
1. For practical purposes, it is increasingly difficult to kill insects with fumigants as the temperature is lowered to 10°C. Below this point, in progression, various species or stages may succumb to low temperature or be weakened by it.
2. Adsorption is the most important physical factor modifying the penetration of fumigants. The amount of gas physically adsorbed increases as the temperature is lowered, and it is necessary to add progressively more fumigant to sustain concentrations free to act on the insects. Furthermore, because of this inverse effect, at low temperatures diffusion of the gas into the material is slower during the treatment, and there is a corresponding decrease in the rate of desorption afterwards.
3. Chemical reaction of the fumigant with some of the fumigated material increases as the temperature is raised. If the residues formed are of significance, it is advisable to conduct the treatment at as low a temperature as possible, with due regard for the handicaps to successful results summarized in paragraphs (1) and (2).
In the light of these three main effects the influence of temperature in different types of fumigation may be considered:
1. With commodities that are easily penetrated and are not highly sorptive, fumigation is practicable at relatively low temperatures with fumigants such as methyl bromide. It will be noted that some of the schedules of recommended treatments at the end of this manual include provision for fumigations at temperatures down to 4°C.
2. Fumigation at temperatures at which the insects are not active may be advantageous in some quarantine treatments. There are two principal reasons for this. Firstly, if seeds or live plants in dormant condition are being fumigated, the risk of injury is reduced by avoiding the possible stimulating effects of higher temperatures on physiological mechanisms. Secondly, if the infesting insects are active fliers, their chances of escape from the material awaiting treatment in a cool environment are greatly reduced.
3. With highly sorptive materials, on the other hand, low temperature fumigation may not be advisable because increased adsorption of the gas by the commodity may interfere with penetration. Also, under some conditions, the material may be hazardous for handling because the adsorbed fumigant is held longer at low temperatures.
EFFECT OF HUMIDITY
From the present knowledge of insect toxicology, it is not possible to make any general statements about the influence of humidity on the susceptibility of insects to fumigants. Variations in response at certain humidities have been observed not only between different species subjected to different fumigants but also between stages of the same species exposed to a single fumigant. However, variations due to humidity are not so important in practice as those due to temperature.
The treatments recommended in this manual are adequate for the range of moisture content and humidity normally encountered.
EFFECT OF CARBON DIOXIDE
Carbon dioxide, in certain concentrations, may stimulate the respiratory movements and opening of spiracles in insects. A number of authors have shown that addition of carbon dioxide to some of the fumigants may increase or accelerate the toxic effect of the gas (Cotton and Young, 1929; Jones, 1938; Kashi and Bond, 1975; Bond and Buckland, 1978). With each fumigant acting on different insects, there seems to be an optimum amount of carbon dioxide needed to provide the best insecticidal results. Excessive amounts of carbon dioxide tend to exclude oxygen from insects and thus interfere with the action of the fumigants.
With certain fumigants, such as ethylene oxide and methyl formate, the addition of carbon dioxide may work to advantage both by reducing the fire or explosion hazards and by increasing the susceptibility of the insects. On the other hand, with fumigants that are nonflammable, the advantages of adding carbon dioxide may be offset by the extra cost and work required to handle the additional weight of containers.
The use of carbon dioxide as a "fumigant" introduced artificially into grain storages or other structures is described in Chapter 11.
Some fumigants can produce paralysing effects on insects that may alter the toxicity of these or other fumigants. In the use of hydrogen cyanide (HCN) against insects, it has been shown that, if certain species are exposed to sublethal concentrations before the full concentration is applied, the resulting fumigation is less effective than one in which the insects are subjected to the full concentration from the very beginning (Lindgren, 1938). A similar protective effect can also occur with the fumigant phosphine if insects are exposed to excessive concentrations during a treatment (Winks, 1974a). Also, insects that have been narcotized by sublethal concentrations of HCN have been found to be protected from lethal treatments with other fumigants, e.g. methyl bromide (Bond, 1961) and phosphine (Bond et al, 1969). This effect has been referred to as "protective stupefaction" or "narcosis".
Although phosphine itself can narcotize insects it does not, however, protect them from the action of methyl bromide as does HCN; in fact, phosphine and methyl bromide can be used together as a "mixture" to enhance the effectiveness of each other (Wohigemuth et al, 1976; Bond, 1978).
From the practical point of view the phenomenon of narcosis is important because it can reduce the effectiveness of certain fumigants. However, steps can be taken to avoid problems of this nature:
1. In fumigations with HCN the maximum concentration attainable from a recommended dosage should be achieved as soon as possible at the beginning of the treatment.
2. HCN should not be applied with other fumigants such as methyl bromide or phosphine, if the maximum toxic effect is to be achieved.
3. Excessive concentrations of phosphine likely to produce a protective narcosis should not be used.
FLUCTUATIONS IN SUSCEPTIBILITY OF INSECTS
It has often been observed that there may be fluctuations in the susceptibility of populations of insects to a given poison. Some of the reasons are known, while others need further clarification. Two important factors are undoubtedly seasonal changes in climate and the effect of nutrition. The susceptibility of insects may be greatly influenced by the quality of the food they consume. It also has been observed with some insects that a certain amount of starvation may make them more, rather than less, resistant to fumigants (Sun, 1946).
In practical work it is well to know that fluctuations in resistance may occur. The alert operator must always be on the lookout for any changed conditions that may necessitate modification of recommended treatments.
COMPARATIVE TOXICITY OF FUMIGANTS
Apart from the influence of the environment, there is a great variation in susceptibility of different species of insects to different fumigants. The successive stages of a given species may also vary greatly in response. Figure 7 illustrates this point. The data were obtained during an extensive study of the usefulness of HCN and methyl bromide for the disinfestation of empty ships (Monro et al, 1952).
Howe and Hole (1966) have shown that these variations in the susceptibility of stages of Sitophilus qranarius (L.), observed under practical conditions, are closely confirmed in laboratory experiments.
A large number of studies have been made under laboratory conditions to determine the relative susceptibility of insects to different fumigants. Table 16 (Chapter 14) shows how fumigants may vary in their toxicity to common species. Bowley and Bell (1981) have reported on the toxicity of twelve fumigants to three species of mites infesting grain.
The treatments recommended here are based on laboratory or field trials that have been confirmed, in many instances, by the results of successful application in practice. Note that all recommended treatments refer to specific insects or their stages or, in some cases, to clearly defined groups of insects. There is, therefore, no guarantee of the success of any attempts to apply a treatment outside the limits given in the recommended schedules.
Many species of insect have the ability to develop resistance to certain insecticides. With fumigants this problem of resistance is a matter of increasing concern; in a global survey of stored grain pests, resistance to both of the major fumigants, phosphine and methyl bromide, was found in a number of insect species (Champ and Dyte, 1976). Collections of 849 strains of insects from 82 countries showed that 20 percent of the insects had some resistance to phosphine and 5 percent to methyl bromide. The highest level of resistance (10-12 times normal) was found in the lesser grain borer Rhyzopertha dominica (F.). It was concluded from this survey that resistance to fumigants was, as yet, limited in extent and often at marginal levels, but that it was of considerable significance as it posed a real threat to the future use of fumigants as control agents.
FIGURE 7. - The relative
susceptibility of different life stages of Sitophilus qranarius
to HCN (dosage 0.72 to 0.84 percent by volume) and methyl bromide
(0.21 to 0.76 percent by volume) during the fumigation of empty
cargo ships. Exposure for 10 to 12 hours, temperature range 3 to
(Monro et al, 1952)
Research in laboratories has shown that a number of destructive stored product insects can develop appreciable resistance to fumigants. Selection of the granary weevil (Sitophilus qranarius) has produced a strain with more than 12fold resistance to methyl bromide (Bond and Upitis, 1976). A strain of the red flour beetle, Tribolium castaneum (Herbst), developed a 10-fold resistance to phosphine in six generations (Winks, 1974b).
There is recent evidence, from field studies in India and Bangladesh, of the development of resistance to phosphine in the Khapra beetle (Borah and Chalal, 1979) and other stored grain pests (Tyler et al, 1983).
Resistance to fumigants is of concern because of the great value of fumigants for pest control and because of the very limited number of materials available. Even low levels of resistance in species of insects that are cosmopolitan and easily transported to other parts of the world could be of serious consequence.
In view of the importance of resistance to fumigation, a brief and simplified account of some features of the problem are given below.
HOW RESISTANCE DEVELOPS
When a population of insects is exposed to an insecticide some individuals are killed more easily than others. The insects that are more difficult to kill may survive after the treatment and produce offspring that are also hard to kill. These insects are said to be more tolerant because they can withstand above-average doses of the poison. If a population is repeatedly treated with the same insecticide and each new generation has increasingly higher tolerance, a "resistant" strain is produced. Resistance is a genetic characteristic that is passed on from one generation to the next.
In the laboratory, resistance is produced by treating a population to kill most of the insects, breeding the tolerant survivors to produce a new generation, re-treating and continuing the process until a resistant strain is obtained. This process is known as selection for resistance. A number of strains of insects with resistance to different fumigants have been produced in this way (Monro et al, 1972; Bond, 1973; Winks, 1974b; Bond and Upitis, 1976).
In the field, resistance to fumigants can develop in the same way. In a grain bin, on a cargo ship or any other place where a resident population of insects is treated over and over again with the same fumigant, resistance might develop. Insects that are not killed may produce new generations with increasingly greater tolerance. Generally, resistance does not develop as readily in wild populations as in the laboratory because the selection process .may be irregular and because they may interbreed with nontreated susceptible insects. However, the fact that resistance has been discovered in wild populations indicates the possibility that further resistance may develop where fumigants are used regularly.
NATURE OF RESISTANCE
Resistance is an inborn characteristic that allows individual insects to tolerate above average doses of a poison. Resistant insects usually are similar in appearance and have the same habits as susceptible insects. Normally, they can only be distinguished by their ability to tolerate excessive concentrations of the fumigant. Tests have been designed for detecting and measuring resistance to fumigants (FAO, 1975; UK, 1980).
An important feature in resistance is the ability to tolerate the effects of more than one poison. Insects that have resistance to one fumigant can, in some cases, also be resistant to other fumigants. This characteristic, known as "cross-resistance" is demonstrated by the data in Table 5. It can be seen that granary weevils selected with methyl bromide were also resistant to several other fumigants, and the levels of cross-resistance were all significant in terms of practical control. Such cross-resistance was not found, however, in insects selected with phosphine (Monro et al, 1972; Kem, 1978) or ethylene dibromide (Bond,1973).
TABLE 5. - RESPONSE OF METHYL BROMIDE - RESISTANT GRANARY WEEVIL TO SIX OTHER FUMIGANTS*
*Dosage in mg/l for 5h, at 25°C required for 50 percent mortality (Monro et al,1961).
TESTING FOR RESISTANCE (FAO, 1975)
For routine monitoring to detect the initial appearance of resistance in wild populations of stored product beetles, it is convenient to use a discriminating dose, which is expected to kill all susceptible specimens. The dose chosen is that corresponding to slightly above the LD(99) 9 obtained from the regression line for susceptible beetles allowing for, in the case of phosphine, what appears to be inherent variability of response. Some discriminating concentrations are given in Table 6. Susceptible reference strains must always be included in discriminating tests.
When using a discriminating test with fumigants it is always advisable to make provision for abnormal concentrations. If a concentration is obtained that is less than the discriminating concentration, this will be revealed by abnormal survival in the susceptible reference strain. Abnormally high concentrations may be revealed by the inclusion in the tests of a reference strain (or species) with slightly greater tolerance to the fumigant than the susceptible reference strain on which the discriminating dose is based, approximately x 1.5 for methyl bromide tests and x 2.5 for phosphine tests. An alternative approach is to use three dosages, one at the discriminating dose, one at the approximate LD(90) level and the other at an equivalent level above the discriminating dose.
In regular monitoring for resistance, it should be detectable even when only a small proportion of resistant individuals is present. A minimum of 100 insects in two batches of 50 should be used per sample.
Limited numbers of insects may not be sufficient to detect low levels of resistance. Therefore, additional samples should be obtained, if possible. If, however, there is suspicion of serious resistance (e.g. from failure of treatments) a test with small numbers (10 to 20) may provide valuable early indication.
The insects are exposed to the discriminating dose for the appropriate period in the usual way. If all of them are dead at the end of the posttreatment holding period, the sample can be classified as "no resistance detectable", and the medium in which they were held is put into a hot-air oven to destroy the culture. On the other hand, the presence of surviving insects at the end of the test should be regarded as prima facie evidence of resistance and investigated further.
The appearance of unaffected insects in a discriminating test could be due to the presence of unusually tolerant individuals from a normal population. Provided that the conditions of exposure, the physiological state of the insects and the dosages are consistent, the probability of a single insect in a batch of 100 being unaffected due to chance is less than 0.1 (e.g. less than once in 10 tests). It is important to determine whether incomplete response is due to such causes or to genuine resistance. This can be checked as follows:
TABLE 6. - NORMAL SUSCEPTIBILITY DATA OBTAINED FOR METHYL BROMIDE AND PHOSPHINE, WITH DISCRIMINATING DOSAGES.
|(Exposure period - 5 hours)
|Sitophilus oryzae (L.)
|S. zeamais Motsch.
|S. qranarius (L.)
|Rhyzopertha dominica (F.)
|Tribolium castaneum (Herbst)
|T. confusum Duv.
|Oryzaephilus surinamensis (L.)
|O. mercator (Fauv.)
(Exposure period - 20 hours)
1. The test can be repeated using further samples from the same field population. The chances of adventitious failure to respond by a single individual in each of successive tests decline progressively (less than 0.01, 0.001, 0.0001 and so on). Survival of two or more indviduals throughout is even less likely. Therefore, the continued appearance of a proportion of unaffected individuals can be considered as proof of resistance.
2. Alternatively, the insects which were unaffected in the discriminating test may be kept and used for breeding a further generation. If their reaction is actually due to resistance, it will be found that a substantially larger proportion of their progeny will fail to respond to the discriminating dose.
When these tests indicate that a population of insects is resistant, then extensive testing should be carried out to determine the degree of resistance present.
WAYS TO AVOID RESISTANCE
Precautions can be taken to reduce the possibility of insects developing resistance to fumigants.
Perhaps the most effective measure involves alternate control practices that do not require chemicals. Good sanitation procedures, proper storage conditions, insect resistant packaging and all other measures that prevent infestations from developing can do much to reduce the need for fumigants. Treatments such as aeration of the commodity, irradiation, temperature extremes, insect pathogens, etc. as listed in Chapter 1 can also be employed.
Where fumigants have to be used on a regular basis, close guard should be kept against control failures. Complete control of all insects in a treatment is the best insurance against resistance.
Periodic checks for resistance should be made in areas that are fumigated regularly. If signs of resistance begin to appear (as indicated either by control failures or through the test procedure) then every effort should be made to eradicate the population. The measures necessary for eradication will vary in different situations; they may involve a number of procedures using both chemical and non-chemical means.
Rotation of fumigants may be effective in some instances, especially if crossresistance is not a problem. For example, methyl bromide might be used at intervals in a control programme that relies mainly on phosphine.
One measure that is not advisable in dealing with resistance problems involves increased dosing. Such practices as doubling the dose of fumigant to achieve an economic level of control can magnify the problem unless complete eradication is assured. Any insects surviving increased doses may develop even higher levels of resistance than would occur with the normally recommended treatment.
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